CN114395530A - Methods for producing autologous T cells for the treatment of B cell malignancies and other cancers and compositions thereof - Google Patents

Abstract

The present invention relates to the field of T cells and provides methods for the production of autologous T cells and compositions thereof for the treatment of B cell malignancies and other cancers. A method of making T cells that express a cell surface receptor that recognizes a specific antigenic moiety on the surface of a target cell, the method comprising enriching a population of lymphocytes; stimulating the population of lymphocytes with one or more T cell stimulators to produce a population of activated T cells, the stimulating being performed in a closed system using a serum-free culture medium; transducing a population of activated T cells with a viral vector comprising a nucleic acid molecule encoding a cell surface receptor, the transduction producing a population of transduced T cells using single cycle transduction, the transduction being performed in a closed system using serum-free culture medium; the transduced T cell population is expanded for a predetermined time to produce an engineered T cell population, the expansion being performed in a closed system using serum-free culture medium. The methods and processes described herein can be completed in a significantly shorter time.

Description

Methods for producing autologous T cells for the treatment of B cell malignancies and other cancers and compositions thereof

Divisional application

The present invention is a divisional application of chinese patent application with application number 201680038907.4 entitled "method for producing autologous T cells for the treatment of B cell malignancies and other cancers and compositions thereof", filed on 4.2.2015.

Technical Field

The present invention relates to the field of T cells and provides methods of making T cells that express cell surface receptors, populations of engineered T cells that express cell surface receptors, pharmaceutical compositions, and methods of making T cells.

Background

The process for producing self-engineered T cells for use in cancer therapy is very time consuming (10-24 days), involves two retroviral transduction cycles, and is poorly suited for commercial applications (see Kochenderfer et al, blood.2012119: 2705-2720; Johnson et al, blood.2009; 114(3): 535-546). Therefore, it is desirable to develop improved methods for T cell manufacturing processes to overcome these limitations.

Disclosure of Invention

According to certain embodiments of the present invention, the present invention provides methods for producing T cells that express cell surface receptors that recognize specific antigenic moieties on the surface of target cells. In certain embodiments, the invention provides methods for making T cells for a cell surface receptor that recognizes a particular antigenic moiety on the surface of a target cell, the method comprising enriching a population of lymphocytes from a donor subject; stimulating the population of lymphocytes with one or more T cell stimulators, thereby producing a population of activated T cells, wherein the stimulation is performed in a closed system using a serum-free culture medium; transducing a population of activated T cells with a viral vector comprising a nucleic acid molecule encoding a cell surface receptor, using single cycle transduction to produce a population of transduced T cells, wherein the transduction is performed in a closed system using serum-free culture medium; and expanding the transduced T cell population for a predetermined time, thereby producing an engineered T cell population, wherein the expanding is performed in a closed system using serum-free culture media. In certain embodiments, the cell surface receptor can be a T Cell Receptor (TCR) or a Chimeric Antigen Receptor (CAR). In certain embodiments, the target cell can be a cancer cell. In certain embodiments, the cancer cell can be a B cell malignancy. In certain embodiments, the cell surface receptor may be an anti-CD19 CAR. In certain embodiments, the anti-CD19 CAR can be a FMC63-28Z CAR or a FMC63-CD828BBZ CAR. In certain embodiments, the one or more T cell stimulating agents may be an anti-CD 3 antibody and IL-2. In certain embodiments, the viral vector may be a retroviral vector. In certain embodiments, the retroviral vector may be a MSGV1 γ -retroviral vector. In certain embodiments, the MSGV1 γ -retroviral vector may be an MSGV-FMC63-28Z or an MSGV-FMC63-CD828BBz γ -retroviral vector. In certain embodiments, the predetermined time for expansion of the transduced T cell population may be 3 days. In certain embodiments, the time from enriching the lymphocyte population to generating the engineered T cells may be 6 days. In certain embodiments, these engineered T cell populations can be used to treat cancer patients. In certain embodiments, the cancer patient and the donor subject may be the same individual. In certain embodiments, the closure system may be a closed bag system. In certain embodiments, the population of cells may comprise naive T cells. In certain embodiments, about 35-43% of the population of engineered T cells may comprise naive T cells. In certain embodiments, at least about 35% of the population of engineered T cells may comprise naive T cells. In certain embodiments, at least about 43% of the population of engineered T cells may comprise naive T cells.

According to a described embodiment of the invention, there is provided a population of T cells expressing a cell surface receptor capable of recognizing a specific antigenic moiety on the surface of a target cell, which population of T cells has been produced by the method of the invention. In certain embodiments, there is provided a method comprising the steps of: enriching a population of lymphocytes from a donor subject; stimulating the population of lymphocytes with one or more T cell stimulators, thereby producing a population of activated T cells, wherein the stimulation is performed in a closed system using a serum-free culture medium; transducing a population of activated T cells with a viral vector comprising a nucleic acid molecule encoding a cell surface receptor, using single cycle transduction to produce a population of transduced T cells, wherein the transduction is performed in a closed system using serum-free culture medium; and expanding the transduced T cell population for a predetermined time, thereby producing an engineered T cell population, wherein the expanding is performed in a closed system using serum-free culture media. In certain embodiments, the engineered T cell population can be any of those described herein.

According to described embodiments of the present invention, pharmaceutical compositions comprising engineered T cell populations are provided. In certain embodiments, the invention provides pharmaceutical compositions comprising populations of engineered T cells described herein. In certain embodiments, the pharmaceutical composition can comprise a therapeutically effective dose of engineered T cells. In certain embodiments, the cell surface receptor can be a T Cell Receptor (TCR) or a Chimeric Antigen Receptor (CAR). In certain embodiments, the CAR can be a FMC63-28Z CAR or a FMC63-CD828BBZ CAR. In certain embodiments, the therapeutically effective dose can be in excess of about 100 to less than about 300 million engineered T cells per kilogram body weight (cells/kg). In certain embodiments, the therapeutically effective dose is about 200 ten thousand engineered T cells per kilogram.

According to described embodiments of the invention, methods for making T cells are provided. The present invention provides a method for producing T cells, comprising the steps of: obtaining a population of lymphocytes; stimulating the population of lymphocytes with one or more T cell stimulators, thereby producing a population of activated T cells, wherein the stimulation is performed in a closed system using a serum-free culture medium; transducing a population of activated T cells with a viral vector comprising a nucleic acid molecule encoding a cell surface receptor, using at least one cycle of transduction to produce a population of transduced T cells, wherein the transduction is performed in a closed system using serum-free culture medium; and expanding the transduced T cell population, thereby producing an engineered T cell population, wherein the expanding is performed in a closed system using serum-free culture media. In certain embodiments, the engineered T cell population can be any of those described herein.

Brief description of the drawings

Figure 1 is a schematic diagram showing a T cell manufacturing process ("modified" process) in certain embodiments of the invention. Since the doubling time of T cells may vary from subject to subject, additional growth times lasting more than 72 hours (i.e. 3-6 days) in the culture bag are considered in case the total number of cells is insufficient to deliver the target dose of interest (see).

Fig. 2 is a schematic diagram showing an improved process compared to a conventionally used process (the "prior" process) according to one embodiment.

FIG. 3 is a bar graph showing improved in-process culture expansion compared to previous processes, according to one embodiment. The Y-axis shows the fold expansion of the cells for each of the 5 runs (x-axis). In large scale engineering runs, culture expansion fold is similar between previous and improved processes.

Figure 4 shows a series of graphs showing T cell phenotypes at day 6 and day 10 in the course of the previous and improved processes for the indicated CD3+ cell phenotype (figure 4A) and CD3+ cell activation (figure 4B) markers, according to one embodiment. The T cell phenotype was comparable between the previous and improved processes, but the cells differentiated to a lesser extent at day 6. Teff ═ effector T cells; tem is an effector memory T cell and Tcm is a central memory T cell.

FIG. 5 shows a series of graphs showing cell phenotype at day 6 of previous and improved processes according to one embodiment.

Fig. 6 is a schematic diagram showing improved cell counts per day during the in-process stimulation, transduction, and amplification phases, according to one embodiment.

FIG. 7 shows the nucleic acid sequence of the MSGV1 gamma-reverse transcription machinery (SEQ ID NO:4), according to one embodiment.

FIG. 8 shows a method for coating a culture bag according to one embodiment

Transduction efficiency as a function of concentration.

Concentration, unit: μ g/mL. Results were measured at day 6 after transduction from two donors in PL07 bags.

FIG. 9 shows transduction efficiencies with and without wash steps, according to one embodiment. In origin PermaLife^TMResults were measured at day 6 after transduction in the bag.

FIG. 10 shows a display according to an embodiment

Concentration pair OpTsizer^TMEffect of transduction efficiency in culture medium.

Concentration, unit: μ g/mL. "open" refers to the condition of transduction, wherein transduction is at

+ 5% human serumIn a titer plate.

Figure 11 shows the activity of transduced T cells assessed by FACS after 4 hours of co-incubation with CD19+ Nalm6 cells as measured by CD107a expression and IFN- γ expression, according to one embodiment. "open" refers to the condition of transduction, wherein transduction is at

+ 5% human serum in a titer plate. Control T represents a reference sample of frozen CAR-positively transduced PBMCs.

Figure 12 shows the product temperature (upper line) and the temperature profile within the program freezer cavity (lower line) for the optimized profile. For simplicity, the curves shown have been truncated so that only critical areas are shown.

Detailed Description

According to described embodiments of the invention, methods or processes for manufacturing T cell preparations are provided that can be used to treat patients suffering from pathological diseases or disorders. In contrast to known methods of T cell production, the methods and processes described herein can be completed in a significantly shorter time, on the order of only 6 days, thereby providing a significant time advantage in bringing these cells into the clinic. Also provided are engineered T cell populations produced using the methods described herein and pharmaceutical compositions thereof.

In certain embodiments, the described methods can be used to make T cells that express cell surface receptors that recognize specific antigenic moieties on the surface of target cells. The cell surface receptor may be a wild-type or recombinant T Cell Receptor (TCR), a Chimeric Antigen Receptor (CAR) or any other surface receptor capable of recognizing an antigenic part associated with a target cell. The form of the antigenic part recognized by the CAR and TCR is slightly different. The CAR has a single chain variable fragment (scFv) as a target binding domain, so that the CAR can be expressed as a single chain protein. This allows the CAR to recognize the original cancer antigen as part of the intact protein on the surface of the target cell. A TCR has two protein chains designed to bind to specific peptides presented by MHC molecules on the surface of certain cells. Since TCRs can recognize peptides in MHC molecules expressed on the surface of target cells, TCRs have the potential to recognize not only cancer antigens presented directly on the surface of cancer cells, but also cancer antigens presented by antigen presenting cells in the tumor, inflammatory and infected microenvironment and secondary lymphoid organs. Antigen presenting cells are the primary immune system cells responsible for amplifying the immune response.

Thus, according to the described embodiments of the invention, the fabricated T cells expressing cell surface receptors can be used to target and kill any target cell, including but not limited to infected cells, injured cells, or dysfunctional cells. Examples of such target cells can include cancer cells, virus-infected cells, bacteria-infected cells, dysfunctional activated inflammatory cells (e.g., inflammatory endothelial cells), and cells involved in a dysfunctional immune response (e.g., cells involved in an autoimmune disease).

In some aspects, the antigenic moiety is associated with a cancer or a cancer cell. Such antigenic moieties may include, but are not limited to, 707-AP (707 alanine proline), AFP (alpha-fetoprotein), ART-4 (adenocarcinoma antigen recognized by T4 cells), BAGE (B antigen; B-catenin/m, B-catenin/mutant), BCMA (B cell maturation antigen), Bcr-abl (breakpoint cluster region-Abelson), CAIX (carbonic anhydrase IX), CD19 (cluster of differentiation 19), CD20 (cluster of differentiation 20), CD22 (cluster of differentiation 22), CD30 (cluster of differentiation 30), CD33 (cluster of differentiation 33), CD44v7/8 (cluster of differentiation 44, exon 7/8), CAMEL (CTL-recognized antigen on melanoma), CAP-1 (carcinoembryonic antigen peptide-1), CASP-8 (caspase-8), CDC27m (cell division cycle 27 mutant), CDK4/m (cyclin-dependent kinase 4 mutant), CEA (carcinoembryonic antigen), CT (cancer/testis (antigen)), Cyp-B (cyclophilin B), DAM (melanoma differentiation antigen), EGFR (epidermal growth factor receptor), EGFRvIII (epidermal growth factor receptor, type III mutant), EGP-2 (epithelial glycoprotein 2), EGP-40 (epithelial glycoprotein 40), Erbb2,3,4 (erythroleukemia virus oncogene homolog-2, -3, -4), ELF2M (elongation factor 2 mutant), ETV6-AML1(Ets variant gene 6/acute myeloid leukemia 1 gene ETS), FBP (folate binding protein), fAchR (fetal acetylcholine receptor), G250 (glycoprotein 250), GAGE sialic acid (antigen), GD2 (bisialoganglioside 2), GD3 (bisganglioside 3), GnT-V (N-acetylglucosamine transferase V), Gp100 (glycoprotein 100kD), HAGE (helicase antigen), HER-2/neu (human epidermal growth factor receptor-2/neurogenic, also known as EGFR2), HLA-A (human leukocyte antigen-A), HPV (human papilloma virus), HSP70-2M (heat shock protein 70-2 mutant), HST-2 (human signet-2), hTERT or hTRT (human telomerase reverse transcriptase), iCE (intestinal carboxylesterase), IL-13R-a2 (interleukin-13 receptor alpha-2 subunit), KIAA0205, KDR (kinase insert domain receptor), kappa-light chain, E (L antigen), LDLR/FUT (low density lipoprotein receptor/GDP-L-trehalose: b-D-enzyme 2-alpha-L fucosyllactose), LeY (Lewis-Y antibody), L1CAM (L1 cell adhesion molecule), MAGE (melanoma antigen), MAGE-A1 (melanoma-associated antigen 1), mesothelin, murine CMV-infected cells, MART-1/Melan-A (melanoma antigen/melanoma antigen A recognized by T cell-1), MC1R (melanocortin 1 receptor), sarcoplasmic globulin/M (sarcoplasmic globulin mutant), MUC1 (mucin 1), MUM-1, -2, -3 (ubiquitous melanoma mutant 1, 2, 3), NA88-A (NAcDNA clone of patient M88), NKG2D (Natural killer cell group 2, member D) ligand, NY-BR-1 (New York mammary differentiation antigen 1), NY-ESO-1 (esophageal squamous cell carcinoma-1), tumor antigen (h5T4), human tumor antigen (H5T4), P15 (protein 15), P190 minor bcr-abl (protein of 190KD bcr-abl), Pml/RARa (promyelocytic leukemia/retinoic acid receptor alpha), PRAME (preferentially expressed melanoma antigen), PSA (prostate specific antigen), PSCA (prostate stem cell antigen), PSMA (prostate specific membrane antigen), RAGE (kidney antigen), RU1 or RU2 (ubiquitous kidney antigen 1 or 2), SAGE (sarcoma antigen), SART-1 or SART-3 (excluding neoplastic squamous antigen 1 or 3), SSX1, -2, -3, -4 (synovial sarcoma X1, -2, -3, -4), TAA (tumor associated antigen), TAG-72 (tumor associated glycoprotein 72), TEL/AML1 (ectopic Ets-family leukemia/acute myeloid leukemia 1), TPI/m (triosephosphate isomerase mutant), TRP-1 (tyrosinase-related protein 1, or gp75), TRP-2 (tyrosinase-related protein 2), TRP-2/INT2 (TRP-2/intron 2), VEGF-R2 (cell vascular endothelial growth factor receptor 2), or WT1 (Wilms' tumor gene).

In some aspects, the cell surface receptor is any TCR that recognizes a specific antigenic moiety on a cancer cell, including, but not limited to, an anti-707-AP TCR, an anti-AFP TCR, an anti-ART-4 TCR, an anti-BAGE TCR, an anti-Bcr-abl TCR, an anti-CAMEL TCR, an anti-CAP-1 TCR, an anti-CASP-8 TCR, an anti-CDC 27M TCR, an anti-CDK 4/M TCR, an anti-CEA TCR, an anti-CT TCR, an anti-Cyp-B TCR, an anti-DAM TCR, an anti-EGFRvIII TCR, an anti-ELF 2M TCR, an anti-ETV 6-AML1 TCR, an anti-G250 TCR, a GAGCR, an anti-GnT-V TCR, an anti-Gp 100 TCR, an anti-HAGE TCR, an anti-HER-2/neu, an anti-HLA-A TCR, an anti-HPV TCR, an anti-70-HSP-2M TCR, an anti-HST-2 TCR, an anti-hTERT TCR, or anti-hTERT TCR, an anti-iCE 0205, an anti-LAGE antigen, anti-LDLR/FUT TCR, anti-MAGE TCR, anti-MART-1/Melan-A TCR, anti-MC 1R TCR, anti-myosin/m TCR, anti-MUC 1 TCR, anti-MUM-1, -2, -3 TCR, anti-NA 88-A TCR, anti-NY-ESO-1 TCR, anti-P15 TCR, anti-P190 minor bcr-abl TCR, anti-Pml/RARa TCR, anti-PRAME TCR, anti-PSA TCR, anti-PSMA TCR, anti-RAGE TCR, anti-RU 1 or anti-RU 2 TCR, anti-SAGE TCR, anti-SART-1 TCR or anti-SART-3 TCR, anti-SSX 1, -2, -3, 4 TCR, anti-TEL/AML 1 TCR, anti-TPI/m TCR, anti-TRP-1 TCR, anti-TRP-2/INT 2, or anti-WT 1 TCR.

In some aspects, the cell surface receptor is any CAR that can be expressed by a T cell and recognizes a specific antigenic moiety on a cancer cell. Certain CARs contain an antigen binding domain (e.g., scFv) and a signal domain (e.g., CD3 zeta chain). Other CARs contain an antigen binding domain (e.g., scFv), a signal domain (e.g., CD3 zeta chain), and a costimulatory domain (e.g., CD 28). Still other CARs contain an antigen binding domain (e.g., scFv), a signal domain (e.g., CD3 zeta chain), and two costimulatory domains (e.g., CD28 and 4-1 BB). Examples of cell surface receptor CARs that may be expressed by T cells produced according to the methods described herein include, but are not limited to, anti-BCMA CARs, anti-CAIX CARs, anti-CD19 CARs, anti-CD 20 CARs, anti-CD 22 CARs, anti-CD 30 CARs, anti-CD 33 CARs, anti-CD 44v7/8 CARs, anti-CEA CARs, anti-EGFRvIII, anti-EGP-2, anti-EGP-40 CARs, anti-Erbb 2,3,4 CARs, anti-FBP CARs, anti-fAchR CARs, anti-GD 2 CARs, anti-GD 3, anti-HER 2/neu CARs, anti-IL-13R-a 2, anti-KDR CARs, anti-kappa-light chain CARs, anti-lecy CARs, anti-L1 CARs, anti-MAGE-a 1 CARs, anti-mesothelin CARs, anti-murine CMV-infected cells, anti-mucc 3 CARs, anti-NKG 2 CAR D ligands, nycar-R CARs, anti-psm CARs-L1-a 4 CARs, anti-PSMA-T a 7375, anti-TAG 72 CARs, anti-tah CARs, anti-tahcs, anti-L2, anti-tts, anti-ttc 3, anti-ttc CAR, anti-ttc 3, anti-ttc, anti-, Or an anti-VEGF-R2 CAR. In one embodiment, the cell surface receptor is any anti-CD19 CAR. In one aspect, the anti-CD19 CAR comprises an extracellular scFv domain, an intracellular and/or transmembrane portion of a CD28 molecule, optionally an extracellular portion of the CD28 molecule, and an intracellular CD3 zeta domain. The anti-CD19 CAR may also comprise additional domains, such as CD8 extracellular and/or transmembrane domains, extracellular immunoglobulin Fc domains (e.g., IgG1, IgG2, IgG3, IgG4), or one or more additional signaling domains, such as 41BB, OX40, CD2, CD16, CD27, CD30, CD40, PD-1, ICOS, LFA-1, IL-2 receptor, fcgamma receptor, or any other co-stimulatory domain with an immunoreceptor tyrosine-based activation motif.

In certain embodiments, the cell surface receptor is an anti-CD19 CAR, e.g., references Kochenderfer et al, J immunoher.2009, 9 months; 32(7): 689-702, "Construction and Pre-clinical Evaluation of Anti-CD19 Chimeric Antigen Receptor (Construction and Pre-clinical Evaluation of an Anti-CD19 clinical Antigen Receptor)" shows FMC63-28Z CAR or FMC63-CD828BBZ CAR, the subject matter of which is incorporated herein by reference to provide methods for constructing vectors for generating T cells expressing FMC63-28Z CAR or FMC63-CD828BBZ CAR.

In other embodiments, the antigenic moiety is associated with a virally infected cell (i.e., a viral antigenic moiety). Such antigenic moieties include, but are not limited to, Epstein-Barr virus (EBV) antigens (e.g., EBNA-1, EBNA-2, EBNA-3, LMP-1, LMP-2), hepatitis A virus antigens (e.g., VP1, VP2, VP3), hepatitis B virus antigens (e.g., HBsAg, HBcAg, HBeAg), hepatitis C virus antigens (e.g., envelope glycoproteins E1 and E2), herpes simplex virus type 1, type 2 or type 8 (HSV1, HSV2 or HSV8), viral antigens (e.g., glycoproteins gB, gC, gE, gG, gH, gI, gJ, gK, gL, gM, UL20, UL32, US43, UL45, 73749 3), Cytomegalovirus (CMV) viral antigens (e.g., gB, gC, gE, gG, gH, gI, gJ, gK, UL 38120, gL, HIV gp 84), envelope-deficient viral antigens (e.g) or other human influenza virus antigens (gp 4642), hemagglutinin (HA) or Neuraminidase (NA)), a measles or mumps virus antigen, a Human Papilloma Virus (HPV) virus antigen (e.g., L1, L2), a parainfluenza virus antigen, a rubella virus antigen, a Respiratory Syncytial Virus (RSV) virus antigen, or a varicella-zostser virus antigen. In such embodiments, the cell surface receptor can be any CAR or any TCR that recognizes any of the aforementioned viral antigens on a cell infected with the target virus.

In other embodiments, the antigenic moiety is associated with a cell having an immune or inflammatory dysfunction. Such antigenic moieties include, but are not limited to, Myelin Basic Protein (MBP), myelin proteolipid protein (PLP), Myelin Oligodendrocyte Glycoprotein (MOG), carcinoembryonic antigen (CEA), proinsulin, glutamate decarboxylase (GAD65, GAD67), Heat Shock Protein (HSP), or any other tissue-specific antigen involved in or associated with pathogenic autoimmune processes.

In certain embodiments, the methods described herein can include the step of enriching a population of lymphocytes obtained from a donor subject. The donor subject may be a cancer patient to be treated with the cell population generated by the methods described herein (i.e., a self-derived donor), or may be an individual who donates a lymphocyte sample to be used to treat a different individual or cancer patient after the cell population is generated by the methods described herein (i.e., a heterologous donor). The lymphocyte population can be obtained from a donor subject by any suitable method used in the art. For example, the lymphocyte population can be obtained by any suitable in vitro method, venipuncture, or other blood collection method for obtaining a blood and/or lymphocyte sample. In one embodiment, the population of lymphocytes is obtained by a method of apheresis.

Enrichment of lymphocyte populations can be achieved by any suitable separation method, including but not limited to the use of a separation medium (e.g., Ficoll-Paque)^TM、RosetteSep^TMHLA whole lymphocyte concentrate mixture, lymphocyte isolation culture medium (LSA) (MP Biomedical) catalog No. 0850494X), cell size, shape or density separation by filtration or elution, immunomagnetic separation (e.g., magnetically activated cell sorting system, MACS), fluorescent separation (e.g., fluorescence activated cell sorting system, FACS) or bead-based column separation.

In some embodiments, the methods described herein may include the steps of: stimulating the lymphocyte population with one or more T cell stimulating agents, thereby producing an activated T cell population. Any combination of one or more suitable T cell stimulating agents may be used to generate an activated T cell population, including, but not limited to, antibodies or functional fragments thereof (e.g., anti-CD 2 antibodies, anti-CD 3 antibodies, anti-CD 28 antibodies or functional fragments thereof) that target T cell stimulating or co-stimulating molecules, T cell cytokines (e.g., any isolated, wild-type or recombinant cytokine, such as interleukin 1(IL-1), interleukin 2(IL-2), interleukin 4(IL-4), interleukin 5(IL-5), interleukin 7(IL-7), interleukin 15(IL-15), tumor necrosis factor alpha (TNF α)) or any other suitable mitogen (e.g., myristoyl phorbol acetate (TPA), Phytohemagglutinin (PHA), concanavalin A (conA), Lipopolysaccharide (LPS), pokeweed mitogen (PWM)) or natural ligands for T cell stimulatory or co-stimulatory molecules.

In some embodiments, the step of stimulating a lymphocyte population described herein may comprise: stimulating the lymphocyte population with one or more T cell stimulators, the stimulation being performed at a predetermined temperature, for a predetermined time, and/or in the presence of a predetermined level of carbon dioxide. In certain embodiments, the predetermined temperature of stimulation is about 34 ℃, about 35 ℃, about 36 ℃, about 37 ℃, about 38 ℃, or about 39 ℃. In certain embodiments, the predetermined temperature of stimulation may be about 34-39 ℃. In certain embodiments, the predetermined temperature of the stimulus may be about 35-37 ℃. In certain embodiments, the predetermined temperature of stimulation may be about 36-38 ℃. In certain embodiments, the predetermined temperature of stimulation may be about 36-37 ℃ and preferably about 37 ℃. In certain embodiments, the step of stimulating the lymphocyte population comprises stimulating the lymphocyte population with one or more T cell stimulators for a predetermined time. In certain embodiments, the predetermined time of stimulation may be about 24-72 hours. In certain embodiments, the predetermined time of stimulation may be about 24-36 hours, about 30-42 hours, about 36-48 hours, about 40-52 hours, about 42-54 hours, about 44-56 hours, about 46-58 hours, about 48-60 hours, about 54-66 hours, or about 60-72 hours. In certain embodiments, the predetermined time of stimulation may be about 48 hours or at least about 48 hours. In certain embodiments, the predetermined time of stimulation may be about 44-52 hours. In certain embodiments, the predetermined time of stimulation may be about 40-44 hours, about 40-48 hours, about 40-52 hours, or about 40-56 hours. In certain embodiments, the step of stimulating the lymphocyte population may comprise stimulating the lymphocyte population with one or more T cell stimulators in the presence of a predetermined level of carbon dioxide. In certain embodiments, the predetermined level of carbon dioxide for stimulation may be about 1.0-10%. In certain embodiments, the predetermined level of carbon dioxide for stimulation may be about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, or about 10.0%. In certain embodiments, the predetermined concentration of carbon dioxide for stimulation may be about 3-7%. In certain embodiments, the predetermined level of predetermined carbon dioxide for stimulation may be about 4-6%. In certain embodiments, the predetermined level of predetermined carbon dioxide for stimulation may be about 4.5-5.5%. In certain embodiments, the predetermined level of predetermined carbon dioxide for stimulation may be about 5%. In some embodiments, the step of stimulating the lymphocyte population may comprise stimulating the lymphocyte population with one or more T cell stimulators at any combination of three conditions, a predetermined temperature, for a predetermined time, and/or in the presence of a predetermined level of carbon dioxide. For example, in one embodiment, the step of stimulating the lymphocyte population may comprise stimulating the lymphocyte population with one or more T cell stimulators at a predetermined temperature of about 36-38 ℃, for a predetermined time of about 44-52 hours, and in the presence of a predetermined level of carbon dioxide of about 4.5-5.5%.

In certain embodiments, the lymphocyte population used in the step of stimulating the lymphocyte population described herein can be a predetermined concentration of lymphocytes. In certain embodiments, the predetermined concentration of lymphocytes can be about 0.1-10.0 x 10⁶Individual cells/mL. In certain embodiments, the predetermined concentration of lymphocytes can be about 0.1-1.0 x 10⁶Individual cell/mL, 1.0-2.0 x 10⁶Individual cell/mL, about 1.0-3.0 x 10⁶Individual cell/mL, about 1.0-4.0 x 10⁶Individual cell/mL, about 1.0-5.0 x 10⁶Individual cell/mL, about 1.0-6.0 x 10⁶Individual cell/mL, about 1.0-7.0 x 10⁶Individual cell/mL, about 1.0-8.0 x 10⁶Individual cell/mL, about 1.0-9.0 x 10⁶Individual cell/mL, or about 1.0-10.0 x 10⁶Individual cells/mL. In certain embodiments, the predetermined concentration of lymphocytes can be about 1.0-2.0 x 10⁶Individual cells/mL. In certain embodiments, the predetermined concentration of lymphocytes can be about 1.0-1.2 x 10⁶Individual cell/mL, about 1.0-1.4X 10⁶Individual cell/mL, about 1.0-1.6X 10⁶Individual cell/mL, about 1.0-1.8X 10⁶Individual cells/mL, or about 1.0-2.0 x 10⁶Individual cells/mL. In certain embodiments, the predetermined concentration of lymphocytes can be at least about 0.1 x 10⁶At least about 1.0 x 10 cells/mL⁶At least about 1.1 x 10 cells/mL⁶At least about 1.2 x 10 cells/mL⁶At least about 1.3 x 10 cells/mL⁶At least about 1.4 x 10 cells/mL⁶At least about 1.5 x 10 cells/mL⁶At least about 1.6 x 10 cells/mL⁶At least about 1.7 x 10 cells/mL⁶At least about 1.8 x 10 cells/mL⁶At least about 1.9 x 10 cells/mL⁶At least about 2.0 x 10 cells/mL⁶Individual cell/mL, at least about 4.0 x 10⁶Individual cell/mL, at least about 6.0 x 10⁶Per mL, at leastAbout 8.0 x 10⁶Individual cells/mL, or at least about 10.0 x 10⁶Individual cells/mL.

In some embodiments, an anti-CD 3 antibody (or functional fragment thereof), an anti-CD 28 antibody (or functional fragment thereof), or a combination of an anti-CD 3 antibody and an anti-CD 28 antibody may be used following the step of stimulating the lymphocyte population. Any soluble or immobilized anti-CD 2, anti-CD 3 and/or anti-CD 28 antibody or functional fragment thereof may be used (e.g., clone OKT3 (anti-CD 3), clone 145-2C11 (anti-CD 3), clone UCHT1 (anti-CD 3), clone L293 (anti-CD 28), clone 15E8 (anti-CD 28)). In some aspects, these antibodies are available from suppliers known in the art, including, but not limited to, Miltenyi Biotec, BD Biosciences (BD Biosciences) (e.g., MACS GMP CD3 pure 1mg/mL, product number 170-. Furthermore, one skilled in the art would understand how to produce anti-CD 3 and/or anti-CD 28 antibodies using standard methods. Any antibodies used in the methods described herein should be produced according to Good Manufacturing Practice (GMP) to comply with relevant institutional guidelines for biological products. In some embodiments, the one or more T cell stimulating agents that may be used in accordance with the step of stimulating a lymphocyte population include antibodies or functional fragments thereof that target T cell stimulating or co-stimulating molecules in the presence of T cell cytokines. In one aspect, the one or more T cell stimulating agents include an anti-CD 3 antibody and IL-2. In certain embodiments, the T cell stimulating agent may comprise an anti-CD 3 antibody at a concentration of about 20ng/mL to 100 ng/mL. In certain embodiments, the concentration of the anti-CD 3 antibody can be about 20ng/mL, about 30ng/mL, about 40ng/mL, about 50ng/mL, about 60ng/mL, about 70ng/mL, about 80ng/mL, about 90ng/mL, or about 100 ng/mL. In certain embodiments, the concentration of the anti-CD 3 antibody can be about 50 ng/mL. In an alternative embodiment, T cell activation is not required. In such embodiments, the method can omit the step of stimulating a lymphocyte population to produce activated T cells, and transduce the lymphocyte population according to the following steps, which can be enriched for T lymphocytes.

In some embodiments, the methods described herein may include the steps of: the activated T cell population is transduced with a viral vector comprising a nucleic acid molecule encoding a cell surface receptor using a single cycle of transduction, thereby generating a transduced T cell population. Several recombinant viruses have been used as viral vectors to deliver genetic material to cells. The viral vector that may be used in accordance with this transduction procedure may be any avid or amphotropic viral vector, including but not limited to recombinant retroviral vectors, recombinant lentiviral vectors, recombinant adenoviral vectors, and recombinant adeno-associated virus (AAV) vectors. In one embodiment, the viral vector used to transduce the activated T cell population is a MSGV1 gamma retroviral vector. In one aspect, such a MSGV1 gamma retroviral vector may comprise the backbone nucleic acid sequence shown in FIG. 6 (SEQ ID NO:4) wherein a nucleic acid fragment comprising a cell surface receptor (e.g., CAR or TCR) sequence is linked to a nucleic acid fragment comprising a MSGV1 gamma retroviral vector sequence. In certain embodiments, the viral vector used to transduce the activated T cell population can be Kochenderfer et al, J immunother.2009 September; 32(7) 689-702 of the MSGV-FMC63-28Z retroviral vector or MSGV-FMC63-CD828BBZ retroviral vector, which is incorporated herein by reference, to provide a method for retroviral vector construction (as provided in the "construction of recombinant retroviral vectors of MSGV-FMC63-28Z and MSGV-FMC63-CD828 BBZ" section of this publication, "materials and methods"). According to one aspect of this embodiment, the viral vector is cultured in a culture medium that is specific for viral vector manufacture. Any suitable culture medium and/or supplement for culturing viral vectors according to the methods described herein can be used in the viral vector inoculum. According to some aspects, the viral vector may then be added to the serum-free culture medium described below during the transduction step.

In certain embodiments, the step of transducing activated T cells can be performed for a predetermined time, at a predetermined temperature, and/or in the presence of a predetermined level of carbon dioxide. In certain embodiments, the predetermined temperature for transduction may be about 34 ℃, about 35 ℃, about 36 ℃, about 37 ℃, about 38 ℃ or about 39 ℃. In certain embodiments, the predetermined temperature for transduction may be about 34-39 ℃. In certain embodiments, the predetermined temperature for transduction may be about 35-37 ℃. In certain embodiments, the predetermined temperature for transduction may be about 36-38 ℃. In certain embodiments, the predetermined temperature for transduction may be about 36-37 ℃, or more preferably 37 ℃. In certain embodiments, the predetermined time of transduction may be about 12-36 hours. In certain embodiments, the predetermined time of transduction may be about 12-16 hours, about 12-20 hours, about 12-24 hours, about 12-28 hours, or about 12-32 hours. In certain embodiments, the predetermined time of transduction may be about 20 hours or at least about 20 hours. In certain embodiments, the predetermined time of transduction may be about 16-24 hours. In certain embodiments, the predetermined time of transduction may be at least about 14 hours, at least about 16 hours, at least about 18 hours, at least about 20 hours, at least about 22 hours, at least about 24 hours, or at least about 26 hours. In some embodiments, the step of transducing the activated T cell population may comprise transducing the activated T cell population with a viral vector at a predetermined level of carbon dioxide. In certain embodiments, the predetermined level of carbon dioxide for transduction may be about 1.0-10%. In certain embodiments, the predetermined level of carbon dioxide for transduction may be about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, or about 10.0%. In certain embodiments, the predetermined level of carbon dioxide for transduction may be about 3-7%. In certain embodiments, the predetermined level of carbon dioxide for transduction may be about 4-6%. In certain embodiments, the predetermined level of carbon dioxide for transduction may be about 4.5-5.5%. In certain embodiments, the predetermined level of carbon dioxide for transduction may be about 5%. In some embodiments, the step of transducing activated T cells described herein can be performed at any combination of three conditions, a predetermined temperature, for a predetermined time, and/or in the presence of a predetermined level of carbon dioxide. For example, in one embodiment, the step of transducing the population of activated T cells may be performed at a predetermined temperature of about 36-38 ℃ for about 16-24 hours and at a predetermined level of carbon dioxide of about 4.5-5.5%.

In some embodiments, the methods described herein may include the steps of: expanding the transduced T cell population for a predetermined time, thereby generating an engineered T cell population. The predetermined time for amplification may be any time suitable for producing: (i) a sufficient number of cells in the engineered T cell population that can be used to administer at least one dose to a patient, (ii) an engineered T cell population having a favorable proportion of naive cells compared to a typical longer course, or (iii) both (i) and (ii). This time will depend on the cell surface receptor expressed by the T cell, the viral vector used, the dose required for therapeutic effect, and other variables. Thus, in some embodiments, the predetermined time for amplification may be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, or more than 21 days. In some aspects, the predetermined time for amplification is shorter than amplification methods known in the art. For example, the predetermined time for amplification can be at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or more than 75% shorter. In one aspect, the predetermined time for amplification is about 3 days. In this aspect, the time from enriching the lymphocyte population to generating the engineered T cells is about 6 days. In certain embodiments, the step of expanding the population of transduced T cells can be performed at a predetermined temperature and/or in the presence of a predetermined level of carbon dioxide. In certain embodiments, the predetermined temperature may be about 34 ℃, about 35 ℃, about 36 ℃, about 37 ℃, about 38 ℃, or about 39 ℃. In certain embodiments, the predetermined temperature may be about 34-39 ℃. In certain embodiments, the predetermined temperature may be about 35-37 ℃. In certain embodiments, the predetermined temperature may be about 36-38 ℃. In certain embodiments, the predetermined temperature may be about 36-37 ℃, or more preferably about 37 ℃. In some embodiments, the step of expanding the transduced T cell population may comprise expanding the transduced T cell population in the presence of a predetermined level of carbon dioxide. In certain embodiments, the predetermined level of carbon dioxide may be about 1.0-10%. In certain embodiments, the predetermined level of carbon dioxide may be about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, or about 10.0%. In certain embodiments, the predetermined level of carbon dioxide may be about 4.5-5.5%. In certain embodiments, the predetermined level of carbon dioxide may be about 5%. In certain embodiments, the predetermined level of carbon dioxide may be about 3.5%, about 4.0%, about 4.5%, about 5.0%, about 5.5%, or about 6.5%. In some embodiments, the step of expanding the population of transduced T cells can be performed at any combination of conditions of a predetermined temperature and/or the presence of a predetermined level of carbon dioxide. For example, in one embodiment, the step of expanding the transduced T cells can include a predetermined temperature of about 36-38 ℃ and the presence of a predetermined level of carbon dioxide of about 4.5-5.5%.

In some aspects, each step of the methods described herein is performed in a closed system. In certain embodiments, the closed system is a closed bag culture system using any suitable cell culture bag (e.g., whirlpool, America)

GMP cell differentiation bag, PermalLife, Origen Biomedical corporation^TMCell culture bags). In some embodiments, the cell culture bags used in the closed bag culture system are all coated with recombinant human fibronectin during the transduction step. In certain embodiments, the cell culture bags used in the closed bag culture system are coated with recombinant human fibronectin fragments during the transduction step. The recombinant human fibronectin fragment may include three functional domains: a central cell binding domain, heparin binding domain II, and CS1 sequence. Recombinant human fibronectin or fragments thereof can be used to target cells by aidingCo-localization of cellular and viral vectors improves the gene efficiency of reverse transcription transduction by immune cells. In certain embodiments, the recombinant human fibronectin fragment is

(Takara Bio, Japan). In certain embodiments, the cell culture bag may be coated with recombinant human fibronectin fragments at a concentration of about 1-60 μ g/mL, preferably about 1-40 μ g/mL. In certain embodiments, the cell culture bag can be coated with recombinant human fibronectin fragments at a concentration of about 1-20 μ g/mL, 20-40 μ g/mL, or 40-60 μ g/mL. In certain embodiments, the cell culture bag can be coated with a recombinant human fibronectin fragment at a concentration of about 1 μ g/mL, about 2 μ g/mL, about 3 μ g/mL, about 4 μ g/mL, about 5 μ g/mL, about 6 μ g/mL, about 7 μ g/mL, about 8 μ g/mL, about 9 μ g/mL, about 10 μ g/mL, about 11 μ g/mL, about 12 μ g/mL, about 13 μ g/mL, about 14 μ g/mL, about 15 μ g/mL, about 16 μ g/mL, about 17 μ g/mL, about 18 μ g/mL, about 19 μ g/mL, or about 20 μ g/mL. In certain embodiments, the cell culture bag can be coated with a recombinant human fibronectin fragment at a concentration of about 2-5 μ g/mL, about 2-10 μ g/mL, about 2-20 μ g/mL, about 2-25 μ g/mL, about 2-30 μ g/mL, about 2-35 μ g/mL, about 2-40 μ g/mL, about 2-50 μ g/mL, or about 2-60 μ g/mL. In certain embodiments, the cell culture bag can be coated with a recombinant human fibronectin fragment at a concentration of at least about 2 μ g/mL, at least about 5 μ g/mL, at least about 10 μ g/mL, at least about 15 μ g/mL, at least about 20 μ g/mL, at least about 25 μ g/mL, at least about 30 μ g/mL, at least about 40 μ g/mL, at least about 50 μ g/mL, or at least about 60 μ g/mL. In certain embodiments, the cell culture bag can be coated with a recombinant human fibronectin fragment at a concentration of at least about 10 μ g/mL. In certain embodiments, human albumin serum (HSA) may be used during the transduction step to close the cell culture bag used in the closed bag culture system. In an alternative embodiment, the cell culture bag is not closed with HSA during the transduction step. In other aspects, one or more of the following steps is performed using a serum-free culture medium without added serum: (a) stimulating a population of lymphocytes; (b) transducing a population of activated T cells; and (c) expandingIncreasing the population of transduced T cells. In another aspect, each of the following steps is performed using serum-free culture media: (a) stimulating a population of lymphocytes; (b) transducing a population of activated T cells; (c) expanding the transduced T cell population. Herein, the term "serum-free medium" or "serum-free culture medium" means that the culture medium used is not supplemented with serum (e.g., human serum or bovine serum). In other words, there is no serum added to the culture medium as a separate and distinct component in order to support viability, activation and growth of the cultured cells. Any suitable culture medium, T cell growth medium, can be used to culture cells in suspension according to the methods described herein. For example, the T cell growth medium may include, but is not limited to, a sterile, low glucose solution containing suitable amounts of buffer, magnesium, calcium, sodium pyruvate, and sodium bicarbonate. In one embodiment, the T cell growth medium is OpTsizer^TM(Life Technologies), but one skilled in the art would understand how to generate similar media. In contrast to typical methods for producing engineered T cells, the methods described herein use culture media without added serum (e.g., human or bovine serum).

According to embodiments described herein, a method of making a T cell expressing a cell surface receptor that recognizes a specific antigenic moiety on the surface of a target cell may comprise the steps of: (1) enriching a population of lymphocytes obtained from a donor subject; (2) stimulating the lymphocytes with one or more T cell stimulators to produce a population of activated T cells, wherein the stimulation is performed in a closed system using serum-free culture medium; (3) transducing a population of activated T cells with a viral vector comprising a nucleic acid molecule encoding a cell surface receptor, using single cycle transduction to produce a population of transduced T cells, wherein the transduction is performed in a closed system using serum-free culture medium; and (4) expanding the transduced T cell population for a predetermined time, thereby producing an engineered T cell population, wherein the expanding is performed in a closed system using serum-free culture medium.

In another embodiment, a method of making a T cell expressing a cell surface receptor that recognizes a specific antigenic moiety on the surface of a target cell may comprise the steps of: (1) enriching a population of lymphocytes obtained from a donor subject; (2) transducing a lymphocyte population with a viral vector comprising a nucleic acid molecule encoding a cell surface receptor, using single-cycle transduction to produce a transduced T cell population, wherein the transduction is performed in a closed system using serum-free culture medium; and (3) expanding the transduced T cell population for a predetermined time, thereby producing an engineered T cell population, wherein the expanding is performed in a closed system using serum-free culture medium.

In one embodiment, these processes or methods may include, but are not limited to: (1) collecting machine-collected blood products from a patient and isolating monocytes in a closed system; (2) stimulating a monocyte population in a closed system with an anti-CD 3 antibody in the presence of IL2, thereby stimulating the growth of T cells; (3) introducing or transducing a novel cell surface receptor gene that allows T cells to recognize specific antigenic moieties on the surface of cancer target cells using a gamma retroviral vector in a closed system; (4) expanding the transduced T cells in a closed system; (5) the expanded autologous T cells are washed and prepared in a closed system for re-administration to a cancer patient. In some aspects, the time for the amplification step is 3 days, such that the entire manufacturing process can be completed in less than 1 week. Steps 2-4, in which T cells are actively growing, are all performed in defined cell culture medium without human serum (i.e. serum-free culture medium). T cells produced by this process exhibit biological activity, can be activated by target antigens on the surface of cancer cells, and in response produce interferon-gamma. Some aspects of the methods described herein include:

this process occurs in a closed system where the possibility of contamination during T cell production has been minimized;

the process is suitable for the production of T cells for clinical use;

propagating the cells in a cell culture medium without human serum;

introducing a receptor gene into a T cell in a closed bag system;

cells can be prepared for clinical use in as short as 6 days; and

the cells exhibit in vivo biological activity.

These aspects provide several differences and/or improvements over the methods currently used in the art, as follows. The use of human serum free cell culture media minimizes the chance of introducing human pathogens from the raw materials during the process and avoids the use of raw materials that are not readily available in the future. Furthermore, this use may support compliance with GMP, since different serum batches require extensive culture testing and release to ensure reproducibility and process stability. T cell growth in serum-free media has been previously reported (Carstens et al, ISCT meeting in San Diego, 2012; Zuliani,2011), but this approach has not previously been incorporated into the production of a process for clinical use to treat cancer, nor has previous work suggested that T cell activation, transduction, and expansion in serum-free media is stable. The use of anti-CD 3 monoclonal antibodies and IL2 to stimulate the T cell population was retained during the improvement expected in the studies described herein. Furthermore, cell culture in closed system bags would provide significant advantages to prevent possible contamination during cell culture and would provide a simple and fast process suitable for cGMP manufacture and product commercialization. The importance of this practical application is significant because many of the processes described in the literature on T cell proliferation are not suitable for large-scale commercial application.

Previously, viral transduction with gamma retroviral vectors was not efficient and an open process called "spin coagulation" was performed in microtiter plates, in which the virus and cells were spun down to a pre-coated layer

On the bottom of the hole. This process is typically repeated twice over consecutive days to maximize transduction efficiency. The transduction step is modified according to some embodiments of the methods described herein so that a pre-coated layer may be used

The bag (rather than the plate) in the closed bag system performs the transduction process and the process only needs to be performed once rather than twice. The methods described herein may also involve cell proliferation in closed system cell culture bags, rather than in open system flasks as previously used in the art. Although some literature has reported transduction in a bag system (Lamers et al, Cytotherapy 2008,10: 406-. Development studies in the described embodiments of the invention show that not only is transduction in a bag system in serum-free culture medium feasible, but that the level of transduction after one transduction is acceptable for future clinical development, and that the amplification time only takes 3 days.

In some embodiments, the engineered T cell populations produced by the methods described above may optionally be cryopreserved for later use of the cells. Accordingly, the invention also provides methods for cryopreserving a population of engineered T cells. Such methods can include the steps of washing and concentrating the population of engineered T cells with a diluent. In some aspects, the diluent is normal saline, 0.9% saline, PlasmaLyte a (PL), 5% dextrose (dextrose)/0.45% NaCl saline solution (D5), Human Serum Albumin (HSA), or a combination thereof. In some aspects, HSA can be added to the washed and concentrated cells to improve the viability and recovery of the thawed cells. In another aspect, the wash is normal saline and the washed and concentrated cells are supplemented with HSA (5%). The method may also include the step of generating a cryopreservation mixture, wherein the cryopreservation mixture comprises the diluted cell population in the diluent and a suitable cryopreservation fluid. In some aspects, the cryopreservation fluid can be any suitable cryopreservation fluid, including but not limited to CryoStor10 (BioLife S (BioLife solutions corporation)Resolution)) was mixed with dilutions of engineered T cells at a ratio of 1:1 or 2: 1. In certain embodiments, HSA may be added to a final concentration of HSA in the cryopreservation mixture of about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, or about 10.0%. In certain embodiments, HSA may be added to a final concentration of HSA in the cryopreservation mixture of about 1-3%, about 1-4%, about 1-5%, about 1-7%, about 2-4%, about 2-5%, about 2-6%, or about 2-7%. In certain embodiments, HSA may be added to a final concentration of about 2.5% HSA in the cryopreservation mixture. For example, in certain embodiments, cryopreservation of a population of engineered T cells can comprise the steps of: washing the cells with 0.9% physiological saline, adding HSA to the washed cells to a final concentration of 5%, and CryoStor^TMCS10 diluted the cells at a 1:1 ratio (resulting in a final concentration of HSA in the final cryopreservation mixture of 2.5%). In some embodiments, the method further comprises the step of freezing the cryopreservation mixture. In one aspect, the cryopreservation mixture is frozen in a program freezer using a given freezing cycle, wherein the cell concentration in the cryopreservation mixture is from about 1e6 to about 1.5e7 cells/mL. The method may further comprise the step of storing the cryopreservation mixture in gaseous liquid nitrogen.

In certain embodiments, the engineered T cell populations produced by the methods described herein can be cryopreserved using a predetermined dose. In certain embodiments, the predetermined dose can be a therapeutically effective dose, which can be any therapeutically effective dose provided below. The predetermined dose of engineered T cells may depend on the cell surface receptors expressed by the T cells (e.g., the affinity and density of the cell surface receptors expressed on the cells), the type of target cells, the nature of the disease or pathological condition being treated, or a combination thereof. In certain embodiments, the cell surface receptor expressed by the engineered T cell may be an anti-CD19 CAR, such as Kochenderfer et al, J immunother, 9 months 2009; 32(7): 689-702, FMC63-28Z CAR or FMC63-CD828BBZ CAR described in "Construction and Pre-clinical Evaluation of Anti-CD19 Chimeric Antigen Receptor" (Construction and Pre-clinical Evaluation of an Anti-CD19 clinical Antigen Receptor), "the subject matter of which is herein incorporated by reference to provide methods for constructing vectors for generating T cells expressing FMC63-28Z CAR or FMC63-CD828BBZ CAR. In certain embodiments, the predetermined dose of engineered T cells expressing FMC63-28Z CAR or FMC63-CD828BBZ CAR can be greater than about 100 to less than about 300 million transduced engineered T cells/kg. In certain embodiments, the predetermined dose of engineered T cells expressing FMC63-28Z CAR or FMC63-CD828BBZ CAR may be greater than about 100 to about 200 ten thousand transduced engineered T cells per kilogram body weight (cells/kg). In certain embodiments, the predetermined dose of engineered T cells expressing FMC63-28Z CAR or FMC63-CD828BBZ CAR may be greater than about 100 to about 200 ten thousand transduced engineered T cells per kilogram body weight (cells/kg). In certain embodiments, the predetermined dose of engineered T cells expressing FMC63-28Z CAR or FMC63-CD828BBZ CAR can be at least about 200 to less than about 300 ten thousand transduced engineered T cells/kg. In certain embodiments, the predetermined dose of engineered T cells expressing FMC63-28Z CAR or FMC63-CD828BBZ CAR may be about 200 ten thousand transduced engineered T cells/kg. In certain embodiments, the predetermined dose of engineered T cells expressing FMC63-28Z CAR or FMC63-CD828BBZ CAR may be at least about 200 ten thousand transduced engineered T cells/kg. In certain embodiments, the predetermined dose of engineered T cells expressing FMC63-28Z CAR or FMC63-CD828BBZ CAR can be about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, or about 290 million transduced engineered T cells/kg. In certain embodiments, a pre-determined dose of about 100 million engineered T cells per kilogram body weight (cells/kg) can be used to cryopreserve a population of engineered T cells. In certain embodiments, a pre-determined dose of about 500000 to about 100 million engineered T cells/kg may be used to cryopreserve a population of engineered T cells. In certain embodiments, a predetermined dose of at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, or at least about 1000 million engineered T cells/kg may be used to cryopreserve a population of engineered T cells. In other aspects, less than 100 ten thousand cells/kg, 200 ten thousand cells/kg, 300 ten thousand cells/kg, 400 ten thousand cells/kg, 500 ten thousand cells/kg, 600 ten thousand cells/kg, 700 ten thousand cells/kg, 800 ten thousand cells/kg, 900 ten thousand cells/kg may be employed, a predetermined dose of 1000, more than 2000, more than 3000, more than 4000, more than 5000, more than 6000, more than 7000, more than 8000, more than 9000 or more than 10000 million cells/kg of a population of engineered T cells is cryopreserved. In certain embodiments, a pre-determined dose of about 100 to about 200 ten thousand engineered T cells/kg may be used to cryopreserve a population of engineered T cells. In other embodiments, the engineered T cell population can be cryopreserved with a predetermined dose of about 100 to about 200, about 100 to about 300, about 100 to about 400, about 100 to about 500, about 100 to about 600, about 100 to about 700, about 100 to about 800, about 100 to about 900, about 100 to about 1000 million cells/kg. In certain embodiments, the predetermined dose of the engineered T cell population may be calculated based on the body weight of the subject. In certain embodiments, the engineered T cell population can be cryopreserved in about 0.5-200mL of cryopreservation medium. In certain embodiments, the engineered T cell population can be cryopreserved in about 0.5mL, about 1.0mL, about 5.0mL, about 10.0mL, about 20mL, about 30mL, about 40mL, about 50mL, about 60mL, about 70mL, about 80mL, about 90mL, or about 100mL of cryopreservation medium. In certain embodiments, the engineered T cell population can be cryopreserved in about 10-30mL, about 10-50mL, about 10-70mL, about 10-90mL, about 50-70mL, about 50-90mL, about 50-110mL, about 50-150mL, or about 100-200mL of cryopreservation medium. In certain embodiments, it is preferred that the engineered T cell population is cryopreserved in about 50-70mL of cryopreservation medium.

The methods described herein are for producing populations of engineered T cells that can be used to treat a subject having a disease or in a pathological state by administering a therapeutically effective amount or dose of the engineered T cells to the subject. Similarly, the methods provided by the invention produce populations of engineered T cells that express cell surface receptors that recognize specific antigenic moieties on the surface of target cells. Pathological conditions that may be treated using the engineered T cells produced by the methods described herein include, but are not limited to, cancer, viral infection, acute or chronic inflammation, autoimmune disease, or any other immune dysfunction. Examples of methods of treating patients With engineered T Cell doses Can Be found in Kochenderfer et al, J Clin oncol.2014 8/25 pi: jco.2014.56.2025 (titled "Chemotherapy-resistant Diffuse Large B-Cell Lymphoma and Refractory B-Cell Malignancies Can Be Effectively Treated using Anti-CD19 Chimeric Antigen Receptor expressed (Chemotherapy-induced difusion target B-Cell Lymphoma and index B-Cell malignanes Can Be e effective expressed With Chemotherapy induced cancer T Cells expression Anti-CD19 Chimeric Antigen Receptor)" and konderfer et al, blood.2012 3/22; 119(12):2709-20 (entitled "B-cell depletion and remission of malignant tumors and cytokine-related toxicity in clinical trials of T cells transduced with anti-CD19 chimeric antigen receptor and the subject matter of a clinical trial of anti-CD19 molecular-anti-organ-receptor-transduced T cells"), the subject matter of which is incorporated herein by reference in its entirety as if fully described herein, thereby providing details on standard practice for treating patients with engineered T cell doses.

According to some embodiments, the engineered T cell population produced by the above methods may comprise one or more subpopulations of cells. In certain embodiments, the one or more subpopulations of cells may include, but are not limited to, naive T cells, effector memory T cells, and/or central memory T cells. As described in example 2 below, one unexpected point is that, when using the methods described herein, in addition to simply shortening the culture time from 10 days or even longer to 6 days, it also resulted in the distribution of more naive T cells, with an increase in initial T cell proportion and a decrease in differentiated effector T cell proportion. In certain embodiments, the engineered T cell population may comprise an initial T cell sub-population. In certain embodiments, at least about 34-43% of the population of engineered T cells may comprise the initial subpopulation of T cells. In certain embodiments, at least about 35% of the population of engineered T cells may comprise a subpopulation of naive T cells. In certain embodiments, at least about 40% of the population of engineered T cells may comprise a subpopulation of naive T cells. In certain embodiments, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, or at least about 44% of the population of engineered T cells may comprise a subpopulation of naive T cells. In certain embodiments, the engineered T cell population may comprise a subpopulation of central memory T cells. In certain embodiments, about 15% or less of the population of engineered T cells may comprise a subpopulation of central memory T cells. In certain embodiments, about 15% or less, about 14% or less, about 13% or less, about 12% or less, about 11% or less of the population of engineered T cells may comprise a subpopulation of central memory T cells.

As used herein, a "cancer" can be any cancer associated with a surface antigen or cancer marker, including, but not limited to, Acute Lymphocytic Leukemia (ALL), Acute Myelogenous Leukemia (AML), adenoid cystic carcinoma, adrenocortical carcinoma, AIDS-related cancer, anal carcinoma, appendiceal carcinoma, astrocytoma, atypical teratoma/rhabdoid tumor, central nervous system, B-cell leukemia, lymphoma or other B-cell malignancy, basal cell carcinoma, cholangiocarcinoma, bladder carcinoma, bone carcinoma, osteosarcoma and malignant fibrous histiocytoma, brain stem glioma, brain tumor, breast carcinoma, bronchial carcinoma, Burkitt's lymphoma, carcinoid tumor, central nervous system carcinoma, cervical carcinoma, chordoma, Chronic Lymphocytic Leukemia (CLL), Chronic Myelogenous Leukemia (CML), chronic myeloproliferative disease, colon carcinoma, Colorectal cancer, craniopharyngeal canal cancer, cutaneous T-cell lymphoma, embryoFetal tumors, central nervous system, endometrial cancer, ependymoma, esophageal cancer, sensorineoblastoma, ewing sarcoma family tumors, extracranial germ cell tumors, extragonadal germ cell tumors, extrahepatic bile duct cancer, eye cancer, fibroblastic histiocytoma of bone, malignancies, and osteosarcoma, gallbladder cancer, stomach (stomach) cancer, gastrointestinal benign tumors, gastrointestinal stromal tumors (GIST), soft tissue sarcomas, germ cell tumors, gestational trophoblastic tumors, gliomas, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular carcinoma (liver cancer), histiocytosis, hodgkin's lymphoma, hypopharynx cancer, intraocular melanoma, islet cell tumors (pancreatic endocrine), kaposi's sarcoma, kidney cancer, langerhans cell histiocytosis, laryngeal cancer, lip and oral cancer, pancreatic cancer, and pancreatic cancer, and pancreatic cancer, and pancreatic cancer, and pancreatic cancer, Hepatoma (primary), Lobular Carcinoma In Situ (LCIS), lung carcinoma, lymphoma, globulinemia, male breast carcinoma, malignant fibrous histiocytoma of the bone and osteosarcoma, medulloblastoma, ependymoma, melanoma, merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer with occult primary mid-line cancer involving the NUT gene, oral cancer, multiple endocrine tumor syndrome, multiple myeloma/plasmacytoma, mycosis fungoides, myelodysplastic syndrome, myelodysplastic/myelodysplastic tumors, Chronic Myelogenous Leukemia (CML), Acute Myelogenous Leukemia (AML), multiple myeloma, myeloproliferative diseases, nasal and sinus cancers, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung carcinoma, oral cancer, pharyngeal cancer, osteosarcoma and malignant fibrous histiocytoma, malignant fibroblastic tumors, cervical cancer, ovarian cancer, pancreatic cancer, multiple papilloma, paraganglioma, sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, mesodifferentiated pineal parenchymal tumor, pineal cytoma and supratentorial primitive neuroectodermal tumor, pituitary tumor, plasmacytoma/multiple myeloma, pleuropulmonary blastoma, pregnancy and breast cancer, primary Central Nervous System (CNS) lymphoma, prostate cancer, rectal cancer, renal cell carcinoma (kidney cancer), renal pelvis and ureteral transitional cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland carcinoma, malignant tumor, melanoma, and malignant lymphomaVenesection tumor, sezary syndrome, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous neck cancer, gastric cancer (gastric carcinoma), supratentorial primitive neuroectodermal tumors, t-cell lymphoma, skin cancer, testicular cancer, throat cancer, thymoma and thymus cancer, thyroid cancer, transitional cell carcinoma of renal pelvis and ureter, trophoblastoma, ureter and renal pelvis cancer, cancer of the urethra, uterine cancer, uterine sarcoma, vaginal cancer, vulval cancer, waldenstrom macroglobulinemia

macrogolulinemia), Wilms Tumor (Wilms Tumor).

In certain aspects, the cancer is a B cell malignancy. Examples of B-cell malignancies include, but are not limited to, non-hodgkin's lymphoma (NHL), diffuse large B-cell lymphoma (DLBCL), small lymphocytic lymphoma (SLL/CLL), Mantle Cell Lymphoma (MCL), Follicular Lymphoma (FL), Marginal Zone Lymphoma (MZL), extralymph node (MALT lymphoma), nodular (monocytic B-cell lymphoma), diffuse large cell splenic lymphoma, B-cell chronic lymphocytic leukemia/lymphoma, burkitt's lymphoma, and lymphoblastic lymphoma.

Herein, a "viral infection" may be an infection caused by any virus that causes a disease or pathological condition in a host. Examples of viral infections that can be treated using the engineered T cells produced by the methods described herein include, but are not limited to, the following viruses: viral infection by Epstein Barr Virus (EBV); viral infections caused by hepatitis a virus, hepatitis b virus or hepatitis c virus; a viral infection caused by herpes simplex virus type 1, herpes simplex virus type 2, or herpes simplex virus type 8, a viral infection caused by Cytomegalovirus (CMV), a viral infection caused by Human Immunodeficiency Virus (HIV), a viral infection caused by influenza virus, a viral infection caused by measles or mumps virus, a viral infection caused by Human Papilloma Virus (HPV), a viral infection caused by parainfluenza virus, a viral infection caused by rubella virus, a viral infection caused by Respiratory Syncytial Virus (RSV), or a viral infection caused by varicella-zostser virus. In some aspects, the viral infection may result or cause the development of cancer in a subject with the viral infection (e.g., HPV infection may result in the development of or be associated with several cancers, including cervical cancer, vulvar cancer, vaginal cancer, penile cancer, anal cancer, oropharyngeal cancer, while HIV infection may result in the development of Kaposi's sarcoma).

Examples of chronic inflammatory diseases, autoimmune diseases or any other immune dysfunction that can be treated with the engineered T cells produced by the methods described herein include, but are not limited to, multiple sclerosis, lupus, and psoriasis.

The terms "treat," "treating," or "therapy" as used herein in relation to a disorder or disease may refer to preventing the disorder or disease, delaying the onset or rate of development of the disorder or disease, reducing the risk of development of the disorder or disease, preventing or delaying development of symptoms associated with the disorder or disease, reducing or terminating symptoms associated with the disorder or disease, producing complete or partial regression of the disorder or disease state, or a combination thereof.

A "therapeutically effective amount" or "therapeutically effective dose" refers to the amount of engineered T cells that produces a desired therapeutic effect in a subject, e.g., by killing target cells to prevent or treat a condition of interest or to alleviate symptoms associated with the condition. The most effective result in terms of therapeutic effect in a given subject will depend on a number of factors including, but not limited to, the characteristics of the engineered T cells (including lifespan, activity, pharmacokinetics, pharmacodynamics, and bioavailability), the subject's physiological condition (including age, sex, disease type and stage, general physical condition, response to a given dose, type of administration), the nature of any pharmaceutically acceptable carrier (carrier) or carriers of any of the ingredients used, and the route of administration. The therapeutically effective dose of the engineered T cells also depends on the cell surface receptors expressed by the T cells (e.g., the affinity and density of the cell surface receptors expressed on the cells), the type of target cell, the nature of the disease or disorder being treated, or a combination thereof. Thus, in some aspects, a therapeutically effective dose of transduced engineered T cells is about 100 to 200 ten thousand transduced engineered T cells per kilogram body weight (cells/kg). Thus, in some aspects, a therapeutically effective dose of transduced engineered T cells is about 100 to 300 million transduced engineered T cells/kg. In certain embodiments, the therapeutically effective dose is about 200 ten thousand transduced engineered T cells/kg. In certain embodiments, the therapeutically effective dose is at least about 200 ten thousand transduced engineered T cells/kg. In certain embodiments, the therapeutically effective dose is at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, ten thousand transduced engineered T cells/kg. In other aspects, the therapeutically effective dose can be less than 100 ten thousand cells/kg, 200 ten thousand cells/kg, 300 ten thousand cells/kg, 400 ten thousand cells/kg, 500 ten thousand cells/kg, 600 ten thousand cells/kg, 700 ten thousand cells/kg, 800 ten thousand cells/kg, 900 ten thousand cells/kg, 1000 ten thousand cells/kg, more than 2000 ten thousand cells/kg, more than 3000 ten thousand cells/kg, more than 4000 ten thousand cells/kg, more than 5000 ten thousand cells/kg, more than 6000 ten thousand cells/kg, more than 7000 ten thousand cells/kg, more than 8000 ten thousand cells/kg, more than 9000 ten thousand cells/kg, or more than 10000 ten thousand cells/kg. In other embodiments, the therapeutically effective dose can be about 100 to about 200 ten thousand cells/kg, about 100 to about 300 ten thousand cells/kg, about 100 to about 400 ten thousand cells/kg, about 100 to about 500 ten thousand cells/kg, about 100 to about 600 ten thousand cells/kg, about 100 to about 700 ten thousand cells/kg, about 100 to about 800 ten thousand cells/kg, about 100 to about 900 ten thousand cells/kg, about 100 to about 1000 ten thousand cells/kg. In some embodiments, the total therapeutically effective dose (number of transduced cells/patient) may be up to about 1e6 transduced cells, about 1e6 to about 1e7 transduced cells, about 1e7-1e8 transduced cells, about 1e8 to about 1e9 transduced cells, about 1e9 to about 1e10 transduced cells, about 1e10 to about 1e11 transduced cells, about 1e11 transduced cells, or more than about 1e11 transduced cells. In one aspect, the therapeutically effective dose can be about 1e8 to about 2e8 transduced cells. One skilled in the clinical and pharmacological arts will be able to determine a therapeutically effective amount by routine experimentation, i.e., by monitoring the subject's response to administration of the compound and adjusting the dosage accordingly. In certain embodiments, the cell surface receptor expressed by the engineered T cell is an anti-CD19 CAR. In certain embodiments, the anti-CD19 CAR can be Kochenderfer et al, J immunoher.2009 September; 32(7) 689-702, the subject matter of FMC63-28Z CAR or FMC63-CD828BBZ CAR, which is herein incorporated by reference, provides methods for constructing vectors for generating T cells expressing FMC63-28Z CAR or FMC63-CD828BBZ CAR. In certain embodiments, a therapeutically effective dose of engineered T cells expressing FMC63-28Z CAR or FMC63-CD828BBZ CAR can be about 100 to about 300 million or less transduced engineered T cells per kg body weight (cells/kg). In certain embodiments, a therapeutically effective dose of engineered T cells expressing FMC63-28Z CAR or FMC63-CD828BBZ CAR can be about 100 million or more to about 200 million transduced engineered T cells per kg body weight (cells/kg). In certain embodiments, a therapeutically effective dose of engineered T cells expressing FMC63-28Z CAR or FMC63-CD828BBZ CAR may be about 200 to about 300 million or less transduced engineered T cells/kg. In certain embodiments, a therapeutically effective dose of engineered T cells expressing a FMC63-28Z CAR or a FMC63-CD828BBZ CAR can be about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, or about 290 million transduced engineered T cells/kg. In certain embodiments, a preferred therapeutically effective dose of engineered T cells expressing FMC63-28Z CAR or FMC63-CD828BBZ CAR is about 200 ten thousand transduced engineered T cells/kg. In certain embodiments, a therapeutically effective dose of engineered T cells expressing FMC63-28Z CAR or FMC63-CD828BBZ CAR is at least about 200 ten thousand transduced engineered T cells/kg.

In some embodiments, a pharmaceutical composition can comprise a population of engineered T cells produced by a method described herein. In certain embodiments, the pharmaceutical composition may also comprise a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier may be a pharmaceutically acceptable material, composition, or vehicle that is involved in carrying or transporting a cell of interest from one tissue, organ, or part of the body to another tissue, organ, or part of the body. For example, the carrier may be a liquid or solid filler, diluent, excipient, solvent or encapsulating material, or a combination thereof. Each component of the carrier must be "pharmaceutically acceptable" in that the component must be compatible with the other components of the formulation. The carrier must also be suitable for contact with any tissue, organ, or part of the body that the carrier may encounter, meaning that the carrier does not carry a risk of toxicity, irritation, allergic response, immunogenicity, or any other complications that may outweigh the therapeutic benefit of the carrier.

The term "about" as used herein means within 5% or 10% of a stated value or range of values.

The following examples are intended to illustrate various embodiments of the present invention. Likewise, the particular embodiments discussed should not be considered limiting to the scope of the invention. For example, although the examples below refer to T cells transduced with an anti-CD19 Chimeric Antigen Receptor (CAR), one skilled in the art will appreciate that the methods described herein may be applied to T cells transduced with all CARs. It will be apparent to those skilled in the art that various equivalents, changes, and modifications may be made, and the present invention includes such equivalent embodiments. In addition, all cited references are incorporated herein by reference in their entirety as if fully set forth herein.

Example 1: preparation of autologous cells modified with in vitro Gene

Fig. 1 provides a schematic diagram of an exemplary T cell production process ("modified" process) consistent with one embodiment. This improved process also includes improvements over the traditionally used T cell producing process (the "previous" process) (see figure 2 showing these improvements) while maintaining T cell product characteristics. In particular, the improved process is a closed process, which unexpectedly avoids the use of serum. And this improved process uses single cycle transduction to produce a population of transduced T cells. Furthermore, using this procedure, cells undergoing a total expansion time of 6 days showed a more naive immunophenotypic profile than cells undergoing a 10 day expansion time. This process enables the reproducible manufacture of products with a targeted number of transduced T cells expressing a Chimeric Antigen Receptor (CAR) such as CD 19; however, these methods are applicable to T cells transduced with any CAR.

In particular, the process is designed to be compatible with organic blood collection products collected using standard mechanical collection equipment and protocols, enrich lymphocytes of a subject's organic blood collection and activate T cells of the subject during a defined culture period in the presence of recombinant IL-2 and anti-CD 3 antibodies, provide an ex vivo culture environment in which T cells can selectively survive and proliferate, transfect T cells of a subject using engineered retroviral vectors to express CD19 chimeric antigen receptors within a consistent range of transfection efficiencies, reduce product-related impurities to consistent levels (product-related impurities include non-T cells in the starting material from the subject), and reduce process-related impurities to consistent levels (process-related impurities include growth media, cytokines, and other process agents).

And (5) collecting mechanical blood collection. Using means such as

Spectra、Spectra

Fenwal^TM

Or equivalent standard mechanical harvesting equipment to collect leukocytes (leukapheresis). This leukapheresis procedure typically produces approximately 200 and 400mL of apheresis product from the patient. The apheresis product may be produced in situ, or optionally transported to a facility at a temperature of 1-10 ℃ to undergo the production process at a different location. Further process steps may be carried out within an ISO 7 cell culture process kit (or similar clean room type environment) as shown in figure 1.

The volume is reduced. When appropriate, using means such as

2 laboratory instruments (Biosafety Inc. (Biosafe SA); Houston, Tex.) or equivalent cell processing instruments perform the modified process volume reduction step and complete the process using standard sterile tubing kits. The volume reduction step is designed to normalize the cell volume to about 120mL, taking into account the variability in the number of cells and the volume of input source material from each subject (about 200-400 mL). If the volume of the mechanical blood collected is less than 120mL, the volume reduction step is not necessary, and the lymphocyte enrichment step can be directly performed on the cells. The volume reduction step is designed to normalize the cell volume received from each subject, preserve monocytes, achieve consistent cell yield and high cell viability, and maintain a closed system, thereby minimizing the risk of contamination.

And (4) enriching the lymphocytes. After the volume reduction step, the separation protocol recommended by the instrument manufacturer (neat cell Program) will be used, for example, using a standard sterile tubing kit

2 or equivalent products on a cell processing apparatus for polysucrose (Ficoll) -based isolation of cells. The lymphocyte enrichment step reduces product-related impurities such as RBCs and granulocytes, enriches and concentrates monocytes, washes and reduces process-related residues such as Ficoll (Ficoll), and prepares cells within a culture medium, thereby providing for cell activation, as well as achieving consistent cell yield and high cell survival. The closed system minimizes environmental contamination.

The process can be carried out at room temperature in the ISO 7 region, and all connections can be made using a sterile pipe welder, as well as within an ISO 5 laminar flow hood.

T cell activation. The T cell activation step may be performed with freshly treated cells from lymphocyte enrichment or with cells previously cryopreserved. If cryopreserved cells are used, the cells should be thawed using protocols that have been developed prior to use.

The T cell activation step selectively activates T cells such that they become transduced with the acceptable retroviral vector, reduces viable populations of all other cell types, achieves consistent cell yields and high T cell survival rates, and maintains a closed system to minimize the risk of contamination.

Wash 1. After the T cell activation step, use is made of

2 or equivalent, the cells are washed with fresh culture medium in a standard sterile kit using protocols developed by the manufacturer. The cells can optionally be concentrated to a final volume of about 100mL in preparation for retroviral vector transduction. The 1 st wash step reduces process related residues such as anti-CD 3 antibodies, used culture media, and cell debris, achieves consistent cell yield and high T cell survival, maintains a closed system to minimize contamination risk, concentrates and delivers sufficient numbers of viable T cells in a small volume suitable for transduction initiation.

Reverse transcription transduction. The activated cells from the 1 st washing step in fresh culture medium were transferred into a cell culture bag (origi Biomedical) PL240 or equivalent device) which had been previously prepared by the following method: first by means of a fluid-like device such as

Recombinant fibronectin or a fragment thereof (Takara Bio, Japan) from Takara Bio Inc. the culture bags were coated and then incubated with a retroviral vector according to the prescribed protocol before introduction into activated cells.

Coating (10. mu.g/mL) was performed at a temperature of 2-8 ℃ for 20. + -.4 hours, washed with dilution buffer, and then thawed with retroviral vector at 37. + -. 1 ℃ and 5. + -. 0.5% CO₂The incubation was continued for about 180-210 minutes. After addition of cells to the culture bag, at 37. + -. 1 ℃ and 5. + -. 0.5% CO₂Transduction was performed for 20. + -.4 hours. The reverse transcription transduction step cultures the activated T cells under controlled conditions and in the presence of a retroviral vector to achieve efficient transduction, achieve consistent cell yield and high cell viability, and maintain a closed system to minimize the risk of contamination.

The 2 nd wash. After the reverse transcription transduction step, use is made of

2 or equivalent, the cells are washed with fresh culture medium in a standard sterile kit using protocols developed by the manufacturer. The cells were concentrated to a final volume of about 100mL in preparation for the expansion step. The 2 nd wash step is designed to reduce process-related impurities such as retroviral vector particles, vector production process residues, spent culture medium and cell debris, achieve consistent cell yield and high cell viability, maintain a closed system to minimize the risk of contamination, and exchange spent culture medium with fresh culture medium having a target number of cells in a specific volume suitable for the start of the amplification step.

T cell expansion. Cells from the 2 nd wash step were aseptically transferred into culture bags (olympic biomedical corporation PL325 or equivalent), diluted with fresh cell culture medium, and incubated at 37 ± 1 ℃ and 5 ± 0.5% CO₂The culture was continued for about 72 hours. Cell density was measured daily from day 5. Since the doubling time of T cells may vary slightly from subject to subject, it is necessary to provide additional growth time in excess of 72 hours (i.e., 3-6 days) if the total cell number is insufficient to deliver the target dose of CAR-positive T cells per kilogram of subject body weight. The T cell expansion step is designed to culture the cells under controlled conditions, thereby producing a sufficient number of transduced cells to provide an effective dose, maintain a closed system to minimize the risk of contamination, and achieve consistent cell yield and high cell survivalAnd (4) rate. One such effective or target dose includes 2 x 10⁶Individual FMC63-28Z CAR-positive or FMC63-CD828BBZ CAR-positive T cells/kg (+ -20%) of subject body weight, generated by transduction using MSGV-FMC63-28Z retroviral vector or MSGV-FMC63-CD828BBZ retroviral vector, respectively, both described in detail in Kochenderfer et al, J immunolther, 9 months 2009; 32(7) 689-702, the subject matter of which is herein incorporated by reference in its entirety as if fully set forth herein.

Wash 3 and concentrate. After the T cell expansion step, use is made of

2 or equivalent, the cells are washed with 0.9% saline in a standard sterile kit using protocols developed by the manufacturer, and after washing the cells are concentrated to a final volume of about 35mL in preparation for formulation and cryopreservation. The 3 rd washing step is designed to reduce process related impurities such as reverse transcription production process residues, spent culture medium and cell debris, achieve consistent cell yield and high cell viability, and maintain a closed system to minimize contamination risk,

once the concentration of cells has been completed and washed in 0.9% saline, the appropriate cell dose can be formulated for preparing the final cryopreserved product. Cells were adapted for cryopreservation and cryopreserved according to the method provided in example 4 below.

Example 2: growth Performance of expanded T cells in cell culture bags

The described embodiments of the invention provide methods for efficient production of engineered autologous T cell therapies within 6 days. The following improvements are achieved in the prior art: the process time is reduced to 6 days instead of 24, 14 or10 days previously used (which reduces the number of tests required for product release (including RCR tests)); improved T cell products, including higher naive T cell ratios, achieving increased potency and effectiveness; a larger number of cells can be used to start the culture to compensate for the shorter manufacturing time; a closed system is used to perform the steps of the method described herein; identifying an unmanned serum culture condition that supports T cell growth; performing single cycle retroviral transduction in a culture bag; cell culture activation and expansion in culture bags instead of flasks; a frozen product is provided. These improvements can be used to develop and commercialize a new engineered peripheral blood T cell therapy (eACT) for the treatment of a variety of cancer indications. The T cells generated from this development protocol maintained the same phenotype and activity profile as the cells propagated by previous methods.

Production of engineered T cells. FIG. 2 shows a schematic of the T cell production process in an improved process with the improvements described in the present invention. Briefly, Peripheral Blood Mononuclear Cells (PBMCs) are obtained from a subject with a B cell malignancy by mechanical harvesting, isolation and parallel (side-by-side) processing using the improved techniques described in the prior art and the present invention. Five studies evaluated all treatment steps by growth at day 6, but did not include final washing and cryopreservation operations. In two additional studies, improved procedures were performed from initial processing of the blood collection material to final formulation and freezing steps, again using organic blood collection products from lymphoma patients.

The enrichment of lymphocytes in machine-harvested blood products (or "samples") was performed by polysucrose (Ficoll) separation of PBMCs using a blocked Sepax2 procedure. The lymphocytes were then plated in serum-free culture medium (OpTsizer) supplemented with developmental prototype supplements (T cell SR Medium supplement, Life Technologies)^TMLife technologies) and closed culture bags, and stimulated with anti-CD 3 antibody and rIL-2 (recombinant IL-2) for 48 hours (days 0-2) for T cell activation. Activated T cells were then washed using a blocked Sepax2 procedure.

On days 2-3 of the manufacturing process, activated T cells were transduced with anti-CD19 CAR using gamma retroviral vectors. This transduction process is accomplished in a closed system as follows. At a concentration of 2-10. mu.g/mL

Coating closed cell culture bags (i.e., origin PermaLife)^TMPL240 culture bag) and then removed

And the bags were washed with buffered saline. The gamma retrovirus was then introduced into the closed system culture bag, followed by an incubation period. The activated T cells were then added directly to the culture bag containing the retroviral vector, followed by overnight incubation at 37 ℃. Will come from

The material of the coated culture bag is removed and placed in a separate cell culture bag for cell expansion. An optional washing step may be added prior to cell expansion. Transduced T cells were expanded in a closed bag system without antibiotics for 3 days (days 3-6). The resulting engineered T cells are then harvested and cryopreserved (cryopreservation is an optional step).

Engineering T cell phenotypes. Engineered T cells were analyzed by Fluorescence Activated Cell Sorting (FACS) for the following purposes: (i) confirming CAR gene expression; (ii) confirming the purity of the T cell population; and (iii) using the T cell subset markers CCR7, CD45RA, CD62L and the functional level markers CD27 and CD28 to determine the phenotype of cells present in the engineered T cell population.

Engineering T cell activity. Engineered T cells were also analyzed using in vitro co-culture bioassay to determine the production of interferon-gamma (IFN- γ) by engineered T cells after co-culture with antigen (Ag) positive (i.e., CD19+) target cells. Engineered T cells were also analyzed by FACS for expression of CD107a and intracellular production of interferon gamma (IFN- γ) following co-culture with antigen (Ag) positive target cells resulting from the engineered T cells.

And (4) growth research. T cell growth and survival were evaluated in each experiment to ensure that cell growth was robust, consistent, and similar to (or superior to) conventional studies.

And (5) freezing and storing. The physiological saline is used on day 6Cells were washed, then HSA was added to 5%, followed by contacting the cells with CryoStor^TM 10(BioLife Solutions^TM) Mixing at a ratio of 1: 1. The cells were then frozen in a program freezer using a defined freezing cycle and then stored in gaseous liquid nitrogen. The results show that the concentration is about 1X 10⁶Cells at-1.5X 10e7/mL can be successfully cryopreserved and thawed by this procedure. The saline containing HSA was as good or better than the other solutions tested (e.g., PL/D5), and HSA improved freeze-thaw recovery.

Viability after cell thawing was assessed by trypan blue exclusion (trypan blue exclusion) and FACS (FACS-based staining for annexin V and 7 AAD). The cells retain their phenotype and biological function after thawing, as measured by IFN- γ.

Results

Growth studies were performed in serum-free culture media along with culture media supplements. In these studies, performance was variable and success was defined as: when the culture medium produced a cell phenotype similar to that produced in a culture medium containing 5% human serum (AIMV). The serum-free culture medium used in the above method produces excellent T cell growth and a cell phenotype similar to that of AIMV. An unexpected observation was that to achieve excellent growth and survival rates, cell densities reached 1.5 x 10⁶at/mL, cells need to be passaged. If cells are not passaged at about this cell concentration, the survival rate decreases. However, in the range of 0.4-1.5e6/mL, the cells grew well, with a doubling time of 24 hours or less when cultured at 37 ℃ in flasks and closed bag systems.

The process of generating engineered T cells, in which a novel receptor gene is introduced into T cells using a gamma retroviral vector, requires that the cells be actively growing so that they can be successfully transduced. In the present invention, T cells are transduced with anti-CD19 CARs, however, the process described herein can be used with any CAR or TCR. The results show that the material can be openedUse of anti-CD 3 antibody and IL2 in an OpTsizer or closed cell culture bag System^TMThe medium stimulates the growth of human T cells. FACS was used to demonstrate that during this stimulation, cells stained with CFSE were at OpTsizer^TMMedia grew equally well as AIMV + 5% human serum. Although T cell growth was seen at other incubation times, the results indicated that in order to do so in the OpTsizer^TMActive growth was obtained in the culture medium, and two days of incubation with anti-CD 3 antibody and IL2 was the best choice.

A number of conditions were evaluated to consider the OpTsizer in a closed bag system^TMTransduction within the medium. A novel aspect of the invention is the OpTsizer developed for use in closed bag systems^TMThe order of specific operational steps to achieve transduction within the medium. The advantage of this procedure is that it is simple to operate, but provides similar transduction frequencies to those of previous use conditions. It is known that steps previously included in the transduction protocol are not necessary (e.g., blocking the coated surface with a protein such as HSA). The transduction frequency is not influenced

The effect of washing the cells after removal from the coated cell culture bag.

Phenotypic analysis of cells at 6 or10 days after the start of the stimulation process indicated that the cells were in the OpTsizer^TMIn media, the cells are substantially similar to cells grown in AIMV media in similar culture bags or commonly used titer plate systems (see FIGS. 4-5). Similarly, in vitro co-culture assays, the OpTsizer was performed in a simple, closed process bag system^TMThe cells produced in the medium were able to produce IFN- γ in response to antigen positive target cells, indicating that the T cells produced by this enhanced process are biologically active.

Table 1 below shows IFN-. gamma.production (pg/ml) at day 6 using the improved procedure described herein.

Samples were from a full-scale engineering run.

UT-untransduced; TD-transduced

Another unexpected observation was that by simply shortening the culture time from 10 days or even longer to the current 6 days, a more naive T cell distribution was produced, while the proportion of primary central memory cells increased and the proportion of differentiated effector T cells decreased (see FIGS. 4-5). This has a beneficial effect on product efficacy and other attributes. Specifically, sufficient cells can be collected from the expanded product after only 3 days of culture. The total time from the start of stimulation to harvest of the expanded, transduced cells was 6 days, which supports about 1-2 x 10⁸Dose of individual CAR-positive cells (figure 6). If a greater number of cells are desired, these cells continue to grow robustly in the culture bag, and the cell culture can be harvested for 10 days or more. Alternatively, a higher number of cells in the starting population may be utilized, thereby generating a larger cell population over a 6 day period.

In addition, two studies were performed at full size with machine blood collected from lymphoma patients, where day 6 cells were further washed with 0.9% saline, formulated in the final product formulation, and cryopreserved. The cell product at day 6 was evaluated for a number of parameters before and after thawing. There was no significant difference in the percentage of CAR positive T cells at 3 days post-thaw compared to levels before freezing, indicating that the cryopreservation protocol was not detrimental to CAR expression. Furthermore, CAR-positive cells continued to show CD 19-specific antigen recognition, as measured by IFN- γ release after co-culture with CD 19-positive target cells. Cell viability upon thawing was 90% and 79% for the two samples tested, respectively.

Example 3: development of transduction conditions in closed systems

Previously, transduction of PBMCs was performed in 6-well plates treated with non-tissue cultures. These plates were incubated at 2-8 ℃ with 10. mu.g/mL

Coating was carried out overnight, or at room temperature for 2 hours. After incubation, removal

The plates were blocked with 2.5% HSA for 30 min, followed by washing with HBSS +5mM HEPES. In a plate-based process, retroviral vectors are applied to coated wells and centrifuged in a centrifuge, followed by removal of about 75% of the viral supernatant, followed by transduction by centrifugation inoculation (centrifugation) to add cells.

In the present invention, three studies were performed to optimize the transduction of PBMCs in closed cell culture bags

And determining whether the transduction was affected by HSA washing and virus supernatant removal. The first experiment was performed in origin PermaLife^TMPL07 culture bag, wherein the cell activation, transduction and expansion are performed in

Vehicle + 5% human serum. 2-40. mu.g/mL were evaluated in PBMCs from three separate donors

Concentration range. At 10. mu.g/mL and 40. mu.g/mL

At concentrations, there was no significant difference between transduction in plates and in culture bags or in the absence of HSA blocking with a confidence interval of 95%. However, at the same confidence interval, will

Reduced to 2. mu.g/mL, or removal of retroviral vectors from the culture bag prior to transduction, appears to reduce transduction efficiency moderately, as shown in Table 2 below。

TABLE 2. compared to transduction performed in plates,

concentration, HSA blocking and retroviral vector removal effects on transduction in cell culture bags

PBMC from two separate donors were used in origin PermaLife^TMPL07 culture bag

A second study conducted in media + 5% human serum confirmed the results obtained in the first study and showed that the transduction efficiency in the culture bag was at

The maximum value is reached at concentrations of 1-20. mu.g/mL (see FIG. 8). Furthermore, the HSA blocking step did not enhance the process or increase the transduction efficiency (see fig. 9). This study also showed that the exclusion of the HSA blocking step did not affect the cell phenotype after transduction (CD45RA/CCR 7).

In a third study, in origin PermaLife^TMPL70 bag pTmizer^TM+ 2.5% supplement or

+ 5% HSA PBMCs from two donors were stimulated for two days. On day 2, cells were washed and transduced with retroviral vectors in PL30 or 6-well plates. Cell concentration during transduction was 0.5 x 10⁶and/mL. On day 3, transduced cells were transferred to T175 flasks (as control) or PL30 culture bags. At day 6 and day 7, the efficacy of the cells was evaluated by co-culture analysis, and CAR expression of the cells was evaluated by FACSAnd a phenotype. FIG. 10 shows

Influence of concentration on the transduction frequency, wherein

At concentrations above 5. mu.g/mL, there was no effect on the frequency of transduction in the bags. FIG. 11 shows that the activity of the cells tested under these conditions was similar when evaluating measurements of cell activation (CD107a expression and IFN-. gamma.production) in response to recognition of the CD19 antigen on target cells. In this case, the transduced cells were incubated with CD19 positive Nalm6 cells for four hours, followed by staining to assess cell surface expression of CD107a and intracellular IFN- γ production.

Thus, according to the method provided by the present invention, the following method of producing a culture bag is supported: the coating has a thickness of 10 mug/mL

The culture bag of (1); using an OpTsizer^TMMedium + 2.5% supplement was transduced in culture bags; the blocking step with HSA had no effect on transduction efficiency or cell potency and phenotype; the T cell products obtained from transduction in the culture bag have a phenotype similar to that of the T cell products obtained from transduction in the plate; retroviral vector removal prior to adding cells to the culture bag during transduction does not increase the overall transduction frequency, but may be reduced.

Example 4: development of cryopreservation step for anti-CD19 CAR-positive T cells manufactured

A series of development studies were performed to determine the optimal conditions for cryopreservation of the anti-CD19 CAR T cells produced. Studies were designed to establish conditions for high survival when thawed, frozen product formulations, and optimal freezing protocols to determine the effect of freeze thawing on cell phenotype and potency. The following analytical methods were used to evaluate performance: cell counting by trypan blue exclusion before and after thawing, annexin staining by FACS after thawing, FACS staining to determine CAR positive T cells and their phenotype (CCR7, CD45RA), growth in cryopreserved cell cultures after thawing, and efficacy assessed by IFN- γ production after co-culture with CD19 positive cells.

Retroviral vector transduced PBMC were used to evaluate the performance measurements described above. In the development study, 3-12X 10 was used in a final cryopreservation volume of 20mL⁶Cryopreserved cells at a concentration range of/mL. Based on the subject body weight and CAR transduction frequency, it is expected that the cell density of the actual clinical product will be within this range. In OriGen CS50 culture bag or AFC

The study was carried out in 20-F bags with no significant difference between the two.

The transduced cells were washed and resuspended in 0.9% saline or

Human Serum Albumin (HSA) may or may not be added to the 1:1 mixture of a and D5 semi-physiological saline (5% glucose/0.45% sodium chloride). These cells are then contacted with

CS10 was mixed at a ratio of 1:1 or 1: 2. In each study, cells were cryopreserved in a program freezer (CRF), stored in gas phase LN2 for more than 2 days, then thawed and evaluated for viability, CAR expression, phenotype and activity. In some experiments, cells were mixed with 80% human AB serum + 20% DMSO at a 1:1 ratio as a control.

When 2.5% HSA (final concentration) was added to the cryopreserved product, cell recovery was enhanced upon thawing (table 3). In addition, cells frozen with HSA maintained higher viability, started to grow faster when placed back into culture, and recovered higher cell viability faster (table 4).

TABLE 3 instant cell recovery of cryopreserved anti-CD19 CAR positive T cells after thawing at different dilutions of cryopreserved

TABLE 4 post-thaw cell recovery and growth of cryopreserved anti-CD19 CAR positive T cells at various dilutions of cryopreserved

Comparison

A study of A/D5 semi-physiological saline and 0.9% saline showed no significant difference in cell recovery upon thawing, cell growth performance or phenotype in the culture after thawing (Table 5). Similarly, use

CS10 dilution of cell phase ratios at a 1:1 ratio

Dilution of cells with CS10 at a ratio of 1:2 did not improve these parameters. In most experiments with a final concentration of HSA in the cryopreserved product of 2.5% and 5% human serum in the T cell diluent, the performance was equal to or superior to cells frozen in a 1:1 ratio of 80% human AB serum/20% DMSO. A decision is therefore made to select a cryopreserved product as follows: cells were washed with 0.9% physiological saline, HSA was added to 5%, and then washed with

CS10 diluted the cells at a ratio of 1: 1.

TABLE 5.0.9% saline comparison

Performance of cells diluted in D5 semi-physiological saline

The refrigeration cycle development was performed on the following selected formulations: 0.45% physiological saline, 2.5% HSA and 50%

CS10, and origin^TMThe final product volume in the CS250 bag was about 50-60 mL. All runs were performed individually, using separate media formulations and volumes for each run. The bags were purged with air and the product temperature was monitored by attaching a thermocouple to the outside surface of the bag. Each bag is placed in a container designed to hold a CS250 bag (e.g., Custom BioGenic Systems)^TMPart number ZC021) and placed on the middle shelf of a freezer rack in a program freezer.

A series of freezing cycles were evaluated to determine 0.45% saline, 2.5% HSA and 50%

The temperature point at which CS10 spontaneously nucleates, thereby aiding in determining when to initiate a temperature peak. After each run, the freezing cycle was modified until a satisfactory sample temperature profile was generated. The criteria for producing a satisfactory sample are that the cold peak offsets the heat of fusion to the greatest extent possible, and that the sample maintains a cooling rate of 1 ℃/min. Table 6 shows the best protocol in a 50mL bag in a program freezer (CRF) and FIG. 12 shows the corresponding product and freezer temperature profiles.

TABLE 6 optimized freezing cycle for anti-CD19 CAR positive T cell product preparation in a program freezer

Step (ii) of	Movement of	Target freezer temperature
			1	Waiting at 4 deg.C	NA
2	Varying at a rate of 1.0 deg.C/min	-23.0℃
			3	Varying at a rate of 30.0 deg.C/min	-75.0℃
4	Change at a rate of 10.0 ℃/min	-28.0℃
			5	Varying at a rate of 1.0 deg.C/min	-40.0℃
6	Change at a rate of 10.0 ℃/min	-90.0℃

Claims (42)

1. A method of making a T cell expressing a cell surface receptor that recognizes a specific antigenic moiety on the surface of a target cell, comprising:

enriching a population of lymphocytes obtained from a donor subject;

stimulating the population of lymphocytes with one or more T cell stimulators, thereby producing a population of activated T cells, wherein the stimulation is performed in a closed system using a serum-free culture medium;

transducing the activated population of T cells with a viral vector comprising a nucleic acid molecule encoding the cell surface receptor using a single cycle transduction to produce a population of transduced T cells, wherein the transduction is performed in a closed system using serum-free culture medium;

expanding the transduced T cell population for a predetermined time to produce an engineered T cell population, wherein the expanding is performed in a closed system using serum-free culture media.

2. The method of claim 1, wherein the cell surface receptor is a T Cell Receptor (TCR) or a Chimeric Antigen Receptor (CAR).

3. The method of claim 1, wherein the target cell is a cancer cell.

4. The method of claim 3, wherein the cancer cell is a B cell malignancy.

5. The method of claim 3, wherein the cell surface receptor is an anti-CD19 CAR.

6. The method of claim 1, wherein the one or more T cell stimulating agents are an anti-CD 3 antibody and IL-2.

7. The method of claim 1, wherein the viral vector is a retroviral vector.

8. The method of claim 7, wherein the retroviral vector is a MSGV1 gamma retroviral vector.

9. The method of claim 1, wherein the predetermined time to expand the transduced T cell population is 3 days.

10. The method of claim 1, wherein the time from enriching the lymphocyte population to producing the engineered T cells is 6 days.

11. The method of claim 10, wherein the population of engineered T cells is used to treat a cancer patient.

12. The method of claim 11, wherein the cancer patient and the donor subject are the same individual.

13. The method of claim 11, wherein the closed system is a closed bag system.

14. The method of claim 1, wherein the population of cells comprises naive T cells.

15. The method of claim 14, wherein about 35-43% of the population of engineered T cells comprise naive T cells.

16. The method of claim 14, wherein at least about 35% of the population of engineered T cells comprise naive T cells.

17. The method of claim 14, wherein at least about 43% of the population of engineered T cells comprise naive T cells.

18. A population of engineered T cells expressing a cell surface receptor that recognizes a specific antigenic moiety on the surface of a target cell, produced by a method comprising the steps of:

enriching a population of lymphocytes from a donor subject;

stimulating the lymphocyte population with one or more T cell stimulating agents to produce an activated T cell population, wherein the stimulation is performed in a closed system using serum-free culture medium;

transducing the activated population of T cells with a viral vector comprising a nucleic acid molecule encoding the cell surface receptor using a single cycle transduction to produce a population of transduced T cells, wherein the transduction is performed in a closed system using serum-free culture medium;

expanding the transduced T cell population for a predetermined time to produce the engineered T cell population, wherein the expanding is performed in a closed system using serum-free culture media.

19. The population of cells of claim 18, wherein the cell surface receptor is a T Cell Receptor (TCR) or a Chimeric Antigen Receptor (CAR).

20. The population of claim 18, wherein the target cells are cancer cells.

21. The population of claim 20, wherein the cancer cells are B-cell malignancies.

22. The population of claim 18, wherein the cell surface receptor is an anti-CD19 CAR.

23. The population of claim 18, wherein the one or more T cell stimulating agents are an anti-CD 3 antibody and IL-2.

24. The population of claim 18, wherein the viral vector is a retroviral vector.

25. The population of claim 24, wherein the retroviral vector is a MSGV1 gamma retroviral vector.

26. The population of claim 18, wherein the predetermined time to expand the population of transduced T cells is 3 days.

27. The population of claim 18, wherein the time from enriching the population of lymphocytes to producing the engineered T cells is 6 days.

28. The population of claim 27, wherein the population of engineered T cells is used to treat a cancer patient.

29. The population of cells of claim 28, wherein the cancer patient and the donor subject are the same individual.

30. The population of claim 18, wherein the closed system is a closed bag system.

31. The cell population of claim 18, wherein the cell population comprises naive T cells.

32. The population of claim 31, wherein about 35-43% of the population of engineered T cells comprise naive T cells.

33. The population of claim 31, wherein at least about 35% of the population of engineered T cells comprise naive T cells.

34. The population of claim 31, wherein at least about 43% of the population of engineered T cells comprise naive T cells.

35. A pharmaceutical composition comprising the population of engineered T cells of any one of claims 18-34.

36. The pharmaceutical composition of claim 35, comprising a therapeutically effective dose of the engineered T cells.

37. The pharmaceutical composition of claim 36, wherein the cell surface receptor is a T Cell Receptor (TCR) or a Chimeric Antigen Receptor (CAR).

38. The pharmaceutical composition of claim 37, wherein the CAR is a FMC63-28Z CAR or a FMC63-CD828BBZ CAR.

39. The pharmaceutical composition of claim 38, wherein the therapeutically effective dose is greater than about 100 to less than about 300 million engineered T cells per kilogram body weight (cells/kg).

40. The pharmaceutical composition of claim 39, wherein the therapeutically effective dose is about 200 ten thousand engineered T cells per kilogram.

41. A method of making a T cell, the method comprising:

(a) obtaining a population of lymphocytes;

(b) stimulating the lymphocyte population with one or more T cell stimulating agents to produce an activated T cell population, wherein the stimulation is performed in a closed system using serum-free culture medium;

(c) transducing the activated population of T cells with a viral vector comprising a nucleic acid molecule encoding the cell surface receptor using at least one cycle of transduction to produce a population of transduced T cells, wherein the transduction is performed in a closed system using serum-free culture medium;

(d) expanding the transduced T cell population to produce an engineered T cell population, wherein the expanding is performed in a closed system using serum-free culture media.

42. The method of claim 41, wherein the cell surface receptor is a T Cell Receptor (TCR) or a Chimeric Antigen Receptor (CAR).

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